Selective Enhancement of REM Sleep in Male Rats through Activation of Melatonin MT1 Receptors Located in the Locus Ceruleus Norepinephrine Neurons

Sleep disorders affect millions of people around the world and have a high comorbidity with psychiatric disorders. While current hypnotics mostly increase non-rapid eye movement sleep (NREMS), drugs acting selectively on enhancing rapid eye movement sleep (REMS) are lacking. This polysomnographic study in male rats showed that the first-in-class selective melatonin MT1 receptor partial agonist UCM871 increases the duration of REMS without affecting that of NREMS. The REMS-promoting effects of UCM871 occurred by inhibiting, in a dose–response manner, the firing activity of the locus ceruleus (LC) norepinephrine (NE) neurons, which express MT1 receptors. The increase of REMS duration and the inhibition of LC-NE neuronal activity by UCM871 were abolished by MT1 pharmacological antagonism and by an adeno-associated viral (AAV) vector, which selectively knocked down MT1 receptors in the LC-NE neurons. In conclusion, MT1 receptor agonism inhibits LC-NE neurons and triggers REMS, thus representing a novel mechanism and target for REMS disorders and/or psychiatric disorders associated with REMS impairments.


Introduction
Sleep disorders represent a major medical problem affecting the health and quality of life of millions of people around the world and are often in comorbidity with psychiatric disorders (Benca et al., 1992), neuropathic pain, and many other medical conditions (Taylor et al., 2007).Sleep is a succession of usually 4-6 cycles per night of non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) episodes lasting ∼90 min (Atkin et al., 2018).Approved medications for sleep disorders include benzodiazepines, Z-compounds, melatonin in a prolonged-release formulation, and more recently orexin antagonists (Atkin et al., 2018).However, these drugs mostly increase NREMS and have either limited effects on enhancing REMS or suppressing REMS (Atkin et al., 2018).REMS is an essential component of sleep, which regulates not only memory consolidation and learning but also emotional processing and movement (Peever and Fuller, 2017).While several receptors activated by monoamines, GABA and glutamate are known to be involved in the regulation of REMS by acting in different and specific brain areas (Aston- Jones and Bloom, 1981;Aston-Jones et al., 2001;Atkin et al., 2018;Osorio-Forero et al., 2022), the role of the melatonin G-protein-coupled receptors (GPCRs) MT 1 and MT 2 is still unknown.
Polysomnography in MT 1 receptor knock-out mice showed a selective reduction of REMS during the sleep/wake cycle, with slight impairments in NREMS, whereas the melatonin MT 2 knock-out mice displayed a decreased duration of NREMS, with the duration of REMS not being affected (Ochoa-Sanchez et al., 2011;Comai et al., 2013).These studies suggested that MT 1 and MT 2 receptors may regulate REMS and NREMS differently, probably due to their unique localization within the brain areas that regulate the sleep/wake cycle (Gobbi and Comai, 2019a,b).Indeed, the pharmacological activation of MT 2 receptors with the selective MT 2 receptor partial agonists UCM765 and UCM924 selectively enhances the duration of NREMS without affecting REMS (Ochoa-Sanchez et al., 2011, 2014;Comai et al., 2013;Gobbi and Comai, 2019a,b), whereas preliminary findings found that the pharmacological activation of MT 1 receptors with the selective MT 1 receptor partial agonist UCM871 enhances REMS (Comai et al., 2017;Lopez-Canul et al., 2019).Until now, a comprehensive study of selective drugs for the MT 1 receptor in sleep function is lacking, and the mechanism by which REMS is regulated by melatonin receptors is still unknown.
Here, we have tested in male rats the first-in-class selective melatonin MT 1 receptor partial agonist named UCM871 (Rivara et al., 2012) and unveiled its mechanism of action in sleep.UCM871 selectively enhances REMS during the 24 h sleep/wake cycle, by decreasing, in a dose-response manner, the firing activity of the locus ceruleus (LC) norepinephrine (NE) neurons, which express MT 1 receptors.Furthermore, using a novel adeno-associated viral (AAV) vector, we selectively knocked down MT 1 receptors on NE-LC neurons, demonstrating that MT 1 receptors in LC-NE neurons are essential for the MT 1 agonism to enhance REMS.

Study design
The overall objective of this study was to investigate the effects of the first-in-class selective melatonin MT 1 receptor partial agonist UCM871 on the sleep/wake cycle and to understand its mechanism of action.Using in vivo EEG/EMG in freely moving animals and single-unit extracellular recordings in anesthetized animals, we first evaluated whether the pharmacological administration of UCM871 promoted sleep and affected sleep architecture throughout the entire 24 h light/ dark cycle and then according to the 12 h light/dark phases.Given the important role of the LC in the modulation of the light/dark cycle and in vigilance states (arousal and REM sleep;Aston-Jones and Bloom, 1981;Carter et al., 2010), we next decided to examine the density and distribution of the MT 1 receptor expression in LC-NE neurons.Due to the expression of MT 1 receptors in LC-NE neurons, we then explored the effect of UCM871 on the spontaneous neuronal firing activity of LC-NE neurons during the light phase, the phase of the day in which we observed the REMS-promoting effects of UCM871.To further demonstrate that the activity of UCM871 upon LC-NE neural firing was MT 1 receptor-mediated, we also pretreated a group of rats with either the MT 2 selective antagonist 4P-PDOT or the non-selective MT 1 /MT 2 luzindole prior to the injection of UCM871.We then hypothesized that MT 1 receptors located in LC-NE neurons were instrumental in increasing REM sleep by UCM871.After knocking down MT 1 receptors in the LC-NE, we tested, in 24 h EEG/EMG recordings, whether the integrity of the MT 1 receptors in LC-NE was required for UCM871 to exert its effects on REMS.Finally, in rats with knockdown of MT 1 receptors in the LC-NE neurons, we investigated whether the electrophysiological effects induced by systemic injection of UCM871 were the result of a local effect on the LC or if they were mediated by other brain regions expressing MT 1 receptors.In in vivo studies, the doses of UCM871 and the sample sizes used were calculated based on our previous experience with melatonin and the congener-selective melatonin MT 2 receptor partial agonists UCM765 and UCM924 in similar experimental models (Ochoa-Sanchez et al., 2011, 2014).Rats were randomly assigned to groups before the experiments, and the number of rats per experimental group is indicated in the figure legends unless otherwise specified.

Animals
The experiments were performed in adult male Sprague Dawley rats (225-340 g; Charles River Laboratories) housed at 22°C with ad libitum access to food and water and maintained under a 12 h light/dark cycle (lights on, 7:30 A.M.; lights off, 7:30 P.M.).Rats were housed in separate cages after the EEG/EMG implant.Experiments were conducted in compliance with the guidelines of and approved by the Animal Care and Welfare Committee of McGill University (protocol number 5253).

Drugs and treatments
The N-[2-[N-methyl-3-(4-phenylbutoxy)anilino]ethyl]acetamide (UCM871; https://pubchem.ncbi.nlm.nih.gov/compound/24763223; 7, 14, and 21 mg/kg; Rivara et al., 2012) was synthesized by the University of Urbino, Italy (Dr.Bedini).Luzindole (10 mg/kg) and 4P-PDOT (4-phenyl-2-propionamidotetralin; 10 mg/kg) were purchased from Sigma-Aldrich.All drugs were dissolved in a vehicle composed of 40% polyethylene glycol 400, 10% ethanol, and 50% saline.The solutions had a pH close to 5.5.The doses of 4P-PDOT and luzindole, as well as UCM871, were chosen based on our previous experiments examining the potential pharmacological activity of these compounds (Ochoa-Sanchez et al., 2014;Lopez-Canul et al., 2015a,b).Drugs were injected subcutaneously (0.5 ml, s.c.) starting at Zeitgeber time (ZT) 10.5 and every 12 h for the EEG/EMG recordings.For electrophysiological recordings, the drugs were administered intravenously between ZT 10.5 and 11.5. Figures 1A and 6A  Implant of EEG/EMG electrodes, EEG/EMG recordings, and EEG/EMG data analysis Implant of EEG/EMG electrodes and the procedures applied for EEG/EMG recording and offline analysis of the three vigilance states were performed following our previous work in rats assessing the effects of melatonin (Ochoa-Sanchez et al., 2014) or the selective MT 2 receptor partial agonist UCM765 (Ochoa-Sanchez et al., 2011) on the sleep/wake cycle.Briefly, anesthetized rats were placed in a stereotaxic frame and implanted with three stainless-steel epidural electrodes for EEG monitoring [one over the parietal cortex on each side and the third (as a reference) in the right prefrontal cortex] and three flexible stainless-steel wire electrodes into the neck muscles for EMG monitoring (two bilaterally and one in the middle).EEG/EMG signals were recorded, subjected to a fast Fourier transform (FFT), and then analyzed offline with the Spike2 software (CED) in 10 s epochs.The three classical vigilance states were assessed based on the cortical EEG and neck EMG activities.EEG power spectra density was also computed in the frequency range of 0-64 Hz.To determine the variations in power spectra induced by the pharmacological treatment with UCM871, we summed up the power densities over the frequency band of 0-25 Hz (total power).The data were then standardized by expressing all power spectral densities at the different 0.5 Hz bin frequency ranges (δ, θ, and σ) as a percentage relative to the total power of the same epoch.
Finally, we also calculated the sleep fragmentation index (SFI) as follows: total number of awakenings from sleep (NREMS and REMS) over the total sleep time in 24 h or 12 h light phase (Morrell et al., 2000).

Single-unit in vivo electrophysiology recording of LC-NE neurons
Rats were anesthetized with chloral hydrate (100 mg/kg, i.p.), placed in a stereotaxic apparatus (David Kopf Instruments), and a catheter was inserted into a tail vein for systemic drug administration.To maintain a full anesthetic state during the experiments, we periodically administered supplemental doses of chloral hydrate (100 mg/kg, i.p.).Anesthesia was confirmed by the absence of a nociceptive reflex reaction to a tail or a paw pinch.
Single-barrel glass micropipettes (Harvard Apparatus), filled with 2% pontamine sky blue dye (Sigma-Aldrich) dissolved in 2 M NaCl (0.5 M, pH 7.5) and with resistances ranging from 2 to 6 MΩ, were used for extracellular recording.Two burr holes with 2.5 mm diameter were drilled bilaterally at the following stereotaxic coordinates: −9.8 mm AP and 1.2 mm ML relative to the bregma (Paxinos and Watson, 2006).LC-NE neurons were recorded lowering the single-barrel glass micropipette at a depth ranging between 6.5 and 8.2 mm (Paxinos and Watson, 2006).Signals were filtered (AC, 0.2-2 kHz), amplified (Bak Electronics, model RP-I), and connected to an oscilloscope (B&K Precision; 20 MHz, model 1522) and an audio system.Signals were digitalized by a CED 1401 interface system, processed online, and analyzed offline by the Spike2 software.LC-NE neurons were identified by their firing patterns (regular firing rate, 0.5-7.5 Hz) and a broad positive action potential (0.8-1.2 ms) frequently characterized by a distinctive notch during the upward phase (Aghajanian and Vandermaelen, 1982;Labonte et al., 2012) and the transient increase of their firing rate characterized by a sequence of bursts in response to a noxious stimulus (pinch in the contralateral hindpaw; Aghajanian and Vandermaelen, 1982).LC-NE burst-firing activity, defined as a train of at least two spikes with an initial interspike interval of <80 ms and a maximum termination interspike interval of 160 ms, was also analyzed according to previously described criteria (Seager et al., 2004).Spontaneous basal firing activity of the LC-NE neurons was recorded for 5 min prior to the intravenous injection of the vehicle followed by cumulative doses of UCM871 (3.5, 7, 10.5, and 14 mg/kg), in 5 min intervals.In the antagonism experiments, luzindole (10 mg/kg, i.v.) or 4P-PDOT (10 mg/kg, i.v.) was injected 5 min after vehicle and 5 min before UCM871 administration.For the animals injected with AAV, the spontaneous firing of LC-NE neurons was assessed 25 d after the viral vector administration, and recorded for 5 min prior to the injection of vehicle or UCM871 (3.5, 7, and 10.5 mg/kg, i.v.).
Once the recordings were terminated, the pontamine sky blue dye was injected iontophoretically by passing a constant positive current of 20 μA for 10 min through the recording pipette to mark the recording site.Then rats were decapitated, and their brains extracted and stored in a freezer at −80°C.Subsequent localization of the labeled site was made by slicing 25-μm-thick brain sections using a cryostat, and the electrode placement was identified with an optical microscope (Olympus U-TV0.5XC-3).All the recording sites were within the LC according to the rat brain atlas (Paxinos and Watson, 2006).
In the vehicle groups, the firing rate was expressed as the percentage of the firing rate after vehicle injection; in the antagonist groups, the firing rate was expressed as the percentage of the firing rate after the antagonist (luzindole or 4P-PDOT) injection.

Immunohistochemistry
To avoid possible confounding effects of the light/dark cycle on melatonin receptor immunolabeling and subcellular localization, we killed all animals between 8:00 and 10:00 A.M. Animals were deeply anesthetized with chloral hydride (100 mg/kg, i.p.) and transcardially perfused with 250 ml of 1× PBS, followed by 500 ml of 4% paraformaldehyde (PFA) in a 0.1 mM phosphate buffer.Their brains were extracted, immersed in the perfusion fixative overnight at 4°C, and transferred to a 30% sucrose solution in 1× PBS for 3 d (4°C).The brains were then immediately snapped frozen in isopentane mixed with dry ice and kept at −80°C.After, the tissue was completely embedded in an OCT compound prior to cryostat sectioning.Coronal sections of 25 µm thickness were obtained using a cryostat (Leica CM3050 S) and immersed in cryoprotective (30% ethylene glycol, 20% glycerol in PBS) solution at −20°C to preserve the tissue.
For single-labeling immunoperoxidase reactivity (n = 5), brain sections obtained from the cryoprotective solution were processed using a free-floating technique, thoroughly washed in 1× PBS solution, immersed in 1% hydrogen peroxide (H 2 O 2 ) for 15 min, and rinsed again in 1× PBS.The sections were then immersed in 0.1% sodium borohydrate in 1× PBS for 20 min and preincubated in a blocking solution (BS) of 10% normal goat serum, 0.5% gelatin, and 0.25% Triton X-100 in 1× PBS for 2 h.For primary antibody incubation, the sections were washed in 1× PBS and incubated at 4°C with rabbit anti-MT 1 (Alomone Labs, 1:1,000 in BS) for 48 h.Next, the sections were rinsed in 1× PBS and then incubated in biotinylated goat anti-rabbit for 2 h and then washed in 1× PBS and incubated in horseradish peroxidaseconjugated streptavidin (1:1,000 in 1× PBS) for 1 h.After rinsing in 1× PBS, the labeling was revealed with a DAB reagent kit (DAB Peroxidase Substrate Kit, catalog #SK-4100, Vector Laboratories) for 2-4 min.This reaction was quickly stopped in distilled water followed by rinsing in 1× PBS.Lastly, sections were mounted and air-dried on glass slides, dehydrated in ethanol (75, 90, 95, and 100%, 5 min each), cleared in xylene (5 min), and coverslipped with DPX (Fluka).
Photomicrographs were taken at low (4×), medium (10×), and high (20×) magnifications with a Leitz Diaplan optical microscope coupled with an Olympus DP21 color digital camera and software (Olympus).Labeled cells were manually counted using ImageJ.A constant outline was used to ensure equally sized areas.
For immunofluorescent colocalization experiments (n = 4), the brain sections were thoroughly washed in 1× PBS and incubated in a blocking solution (BS) of PBST (0.25% Triton X-100 in 1× PBS) with 2% normal goat serum and 2% normal donkey serum for 2 h.For primary antibody binding, the sections were washed in 1× PBS and incubated at 4°C in rabbit anti-MT 1 (Alomone Labs, 1:1,000 in BS) and chicken anti-TH (Abcam, 1:1,000 in BS) for 48 h.Following a wash in 1× PBS, the sections were incubated with Alexa Fluor 555 donkey anti-rabbit IgG secondary antibody (Invitrogen, 1:200 in BS) and goat anti-chicken IgY fluorescein conjugate secondary antibody (Invitrogen, 1:1,000) for 2 h at room temperature.Finally, the sections were rinsed in 1× PBS, mounted on gelatin-coated glass slides to air-dry, and coverslipped with Fluoroshield mounting medium with DAPI (Abcam).
Z-stacks of immunofluorescent images were taken under a Zeiss LSM710 confocal microscope at 20× magnification and processed with ZEISS ZEN Microscopy Software (Carl Zeiss).For quantification, at least three sections per animal were blindly analyzed by multiple operators.Quantification of positive cells was performed with the Fiji Cell Counter plugin.To visualize cells expressing AAV-hrMT 1 (EGFP) in LC-NE neurons, we incubated the slices overnight at 4°C in chicken anti-TH (Abcam, catalog #ab76442, 1:1,000 in BS) and rabbit anti-GFP (Abcam, catalog #ab290, 1:1,000 in BS).After primary antibody incubation, the sections were washed in PBS and then incubated with a secondary antibody anti-rabbit Alexa Fluor (Invitrogen, 1:200 in BS) and anti-chicken IgY H + L (Invitrogen, 1:1,000 in BS) for 2 h at room temperature.To determine cell-specific expression levels of AAV-hrMT 1 , we performed colocalization with the semiquantitative analysis with the JACoP provided Pearson's and Mander's overlap coefficients (http://rsb.info.nih.gov/ij/plugins/track/jacop.html) of the average of three slices per animal (n = 4).M1 signifies the correlation of EYFP + overlapping TH + , while M2 indicates the overlap coefficient of TH + to EYFP + .

Viral vector production
Design of the miR-E embedded shRNA expression cassettes.Briefly, the single-stranded vector vMLC(a)-rh10 (Penn Vector Core) was produced using the custom-made AAV vector plasmid pMLC 2 .The AAV helper plasmid (Addgene #112866) for the production of the AAV serotype rh10 is described in Pillay et al. (2017).vMLC 2 -rh10 induces the constitutive expression of four different human microRNA-30-based (miR-E) (Fellmann et al., 2013) short/small hairpin (sh) RNAs directed against Rattus norvegicus melatonin MT 1 receptor mRNA (NM_053676.2) and the enhanced green fluorescent protein (EGFP).Under the transcriptional control of the artificially eight times repeated cis-regulatory element PRS2 [Phox2a/Phox2b response site, identified in the human dopamine beta-hydroxylase (hDBH) promoter] combined with a minimal hDBH promoter fragment (containing a TATA box and transcription start site), it confers selective expression in noradrenergic and adrenergic neurons.Electronic sequences (TL712063VC, TL712063VA, TL712063VD, TL712063VB; OriGene) were used to create the four miR-E sequences shrMT 1 (1-4) according to Chang et al. (2013).With up to two mismatches, no target sequences of vMLC 2 -rh10 other than that of NM_053676.2were identified in the rat exome.pMLC2 was constructed using the GeneArt Gene Synthesis product and an AAV vector backbone provided by the facility (Neuroscience Center Zurich).The AAV vector plasmid pMLC 2 was amplified using multiple-deletion series (MDS) strain 42 bacteria (LowMut ΔrecA; Scarab Genomics) in combination with anion exchange columns (Macherey-Nagel, NucleoBond Xtra anion exchange columns), plasmid pMLC 2 was then characterized by Sanger DNA sequencing and restriction endonuclease analysis and dissolved in deionized and sterile water.The corresponding control AAV vector plasmid (AAVrh10/ 2-EGFP) was constructed identically with the difference that the short/ small hairpin (sh) is nonsilencing (NS) RNAs.
The identity of encapsidated genomes was verified and confirmed by Sanger DNA sequencing of amplicons produced from genomic AAV vector DNA templates (identity check).
Titers of AAV shr MT 1 vectors, as determined by quantitative PCR, were over 1 × 10 E12 viral genomes (vg)/ml.Single-use aliquots of AAVshr MT 1 and scrambled were stored at −80°C until surgery.
Viral vector administration.AAV injections (shrMT 1 and EGFP) were made in adult rats (200-250 g) anesthetized with 2-5% isofluorane and immobilized on a stereotaxic frame (David Kopf Instruments); two burr holes with 1 mm diameter were drilled bilaterally.A single craniotomy was performed and a Hamilton syringe fitted with a 28-gauge needle was used to place a viral bolus (1 µl) at the following coordinates: 9.8 mm AP, 1.2/−1.2mm ML, relative to the bregma, and −7 mm DV (Paxinos and Watson, 2006).The needle was connected to the drug-filled Hamilton syringe and was left in place for 5 min postinfusion to allow for diffusion of the virus away from the injection site.Next, 1 µl of viral construct or control vector was infused manually at a rate of 0.2 µl/min, followed by 5 min rest period to prevent backflow.Animals were allowed a recovery period of 26 d before use in experiments.Animals with the AAV injection were separated into two different groups: one for electrophysiology and the other implanted with EEG/EMG electrodes, for the posterior sleep/wake cycle analysis.
Quantitative RT-PCR.To validate the effect of the viral vector, we performed quantitative RT-PCR in shr MT 1 (n = 5) and in EGFP (n = 5) injected animals.For this, both LC from each animal were collected between ZT 0.5 and ZT 2.5 and pulled together, and total RNA was extracted from the tissue using the RNA isolation kit RNeasy Lipid Tissue (#1023539, Qiagen) according to the manufacturer's instructions.RNA concentration and purity were determined by UV spectroscopy at 260 and 280 nm.Single-stranded cDNA synthesis was performed from 800 ng of total RNA using the SuperScript VILO cDNA Synthesis Kit (#11754050, Invitrogen) following the manufacturer's instructions.Quantitative real-time PCR was performed using the TaqMan method (Applied Biosystems) with TaqMan Fast Advanced Master Mix (#4444556, Applied Biosystems) and a combination of primers and probes MT 1 #4331182; β-actin (ACTB) #4331182; and POLR2A #4331182 (Thermo Fisher Scientific).Real-time PCR was performed on the QuantStudio 7 Flex system for 40 cycles at 95 and 60°C.The level of expression was determined by converting the cycle threshold (Ct) values using the 2 −ΔΔCt method [with ΔΔCt = ΔCt (mRNA test) -ΔCt (mRNA control AAVrh10/2-EGFP)] to express the relative quantification of each mRNA level in comparison with the level of control AAVrh10/2-EGFP mRNA.
Pharmacokinetic (PK) studies of UCM871.PK studies were conducted measuring the levels of UCM871 in the plasma of the rats after the subcutaneous injection of 7 mg/kg UCM871 dissolved in a vehicle composed of 40% polyethylene glycol 400, 10% ethanol, and 50% saline.The following time points were assessed: 30, 60, 120, and 240 min.Three rats per time point were examined.The pharmacokinetics of UCM871 were described using a noncompartmental model (PK Solver 2.0).The analyses were performed on a TSQ Quantum Ultra triple quadrupole from Thermo Fisher Scientific coupled to a Surveyor series LC stack (Thermo Fisher Scientific) using UCM765 (Ochoa-Sanchez et al., 2011) as internal standard dissolved in LC-MS grade acetonitrile (Thermo Fisher Scientific).The column used was a Kinetex C18, 2.1 × 50 mm, 2 mm (Phenomenex).The eluents were Milli-Q water containing 0.1% formic acid and acetonitrile.The gradient started with 20% organic up to 95% in 1.5 min, held at 95% for 1 min for a total run time of 5 min.The flow was set at 0.350 ml/min and the injection volume at 2 µl.Mass spectra were acquired by electrospray in positive mode using multiple reaction monitoring (MRM) for each compound (UCM871 and UCM765).
UCM871 determination in rat plasma.An aliquot of 30 µl of plasma was protein precipitated with 300 µl of internal standard solution on ice.The mixture was then vortexed for 1 min and centrifuged at 4,000 rpm for 10 min at 4°C.Next, 80 µl of supernatant was mixed with 160 µl of water containing 0.1% formic acid.The mixture was vortexed and submitted to LC-MS/MS analysis.

Statistical analysis
Data analysis was performed using Prism 8 (GraphPad) software.Data were tested for normality using the D'Agostino-Pearson's test.When examining the effect of UCM871 on the 24 h sleep/wake parameters and electrophysiology, one-way ANOVA for repeated measures was performed.For light/dark experiments, two-way ANOVA or two-way ANOVA for repeated measures was applied.Two-tailed paired t test was used for immunoreactivity and PCR experiments and to assess the effects of viral vector infection.Post hoc analyses were performed using the Bonferroni's comparisons.All data are expressed as mean ± SEM. p < 0.05 was considered significant.

Pharmacokinetic properties of UCM871
Using an LC-MS/MS protocol, we determined the concentration of UCM871 in the plasma of three rats after 30, 60, 120, and 240 min (7 mg/kg, s.c.).Thirty minutes after administration, the average plasma concentration of the drug was 132.00 ng/ml, at 60 min it increased to 169.07 ng/ml, at 120 min it decreased to 88.95 ng/ml, and at 240 min it further decreased to 85.45 ng/ml.Using the PK Solver 2.0 software and a non-compartmental model, we found that the half-life of UCM871 was 210.2 min and its Cmax was 169.07 ng/ml.UCM871 selectively increases the duration of REMS during the 24 h light/dark cycle Figure 1D.1-D.3 illustrates the dose-response effects of UCM871 upon the duration of REMS and NREMS and wakefulness during the 24 h light/dark cycle, respectively.UCM871 increased the duration of REMS (Fig. 1D.1;F 3,38 = 4.45, p = 0.009, one-way ANOVA) at a dose of 14 mg/kg (p = 0.02) and 21 mg/kg (p < 0.006) compared with the vehicle.No significant effects of UCM871 on the duration of NREMS (Fig. 1D.2;F 3,38 = 0.37, p = 0.78, one-way ANOVA) and wakefulness (Fig. 1D.3;F 3,38 = 1.30, p = 0.28, one-way ANOVA) were observed.
Power spectra of REMS after treatment with vehicle and different doses of UCM871 during the light and dark phases are reported in Figure 2, D.1 and D.2, respectively.

Melatonin MT 1 receptors are expressed in LC-NE neurons
The specificity of the MT 1 antibodies was first assessed using Western blot analyses.In rat LC homogenates, these antibodies revealed a single band in ∼40 kDa (Figure 3B), corresponding to the molecular weight of the mammalian MT 1 receptor (Audinot et al., 2008).Figure 3A shows the tissue section corresponding to the LC extracted for the Western blotting technique.No bands were detected with the immunogen peptide (Fig. 3B, right panel), suggesting that the protein recognized by the antibodies is the MT 1 receptor.Then, using these antibodies, we found MT 1 -positive immunostaining at the level of the LC (Fig. 3C).Because the MT 1 receptor was localized in the LC, although it seems less abundant in the ventral part of the LC, we then examined whether the MT 1 receptors colocalize within NE-positive neurons in the LC, using immunofluorescence.As shown in Figure 3D, neurons positive for tyrosine hydroxylase (TH) (green) and MT 1 -positive neurons (red) exhibited high colocalization.Interestingly, while a small population of LC neuronal cells was exclusively immunoreactive with MT 1 (14.6%) and another one was exclusively immunoreactive with NE positive (27.4%), most LC neurons (57.9%) exhibited a colocalization, showing a TH-like and MT 1 -like immunoreactivity (Fig. 3E).
ANOVA for repeated measures).These data indicate that UCM871 was acting on the LC-NE neurons through an MT 1 receptormediated mechanism, since the MT 1 /MT 2 antagonist luzindole, but not the MT 2 antagonist 4P-PDOT, blocked the effects of UCM871.
In vivo transfection with shRNA by local delivery of an AAV vector targeting MT 1 receptors in NE-LC neurons We constructed an adeno-associated virus (AAV) vector serotype 10/2 encoding the EGFP and expressing four different shrMT 1 RNAs under the control of the artificial promoter PRS (8×) with hDBH promoter (Fig. 5) to selectively target and silence the expression of MT 1 receptors only in NE neurons.We then injected the vector AAVrh10/2-shrMT 1 -EGFP or the vector AAVrh10/2-EGFP as control bilaterally into the LC of adult rats.The specificity of expression was evaluated 4 weeks postinjection (Fig. 6A), since AAV vectors have been shown to mediate robust transgene expression in most tissues (Hordeaux et al., 2015).To assess the in vivo transfection efficiency of mRNA by AAV vectors, we evaluated positive transfection by immunofluorescence (anti-GFP) in LC-NE neurons and by qRT-PCR.To visualize the expression of the AAV-hrMT 1 viral vector, 26 d after injection, we analyzed the transgene expression by confocal microscopy of coronal sections which were immunostained against TH (to visualize endogenous noradrenergic neurons in the LC) and EGFP (to enhance the signal originating from AAV expression).Nuclei adjacent to the LC and to the 4th ventricle were also stained to rule out possible spilling of the virus from the injection site (Fig. 6B).In the solitary tractus (NST), the prepositus nucleus/mediovestibular nucleus magnocellular (Pr/MVeMC), and the periaqueductal area (PAG), only a few TH + neurons were found, in keeping with the paucity of NE soma in non-LC areas (Svensson, 1987;Berridge and Waterhouse, 2003).A colabeling was observed in only very few neurons, mostly in the area corresponding to the mesencephalic trigeminal nucleus, which contains also TH + neurons.On the Figure 5. Map of the expression of the AAV vector plasmid pMLC 2 gene cassette.Each plasmid carries one expression cassette containing two sequences of AAV ITRs from serotype 2 (purple), one hDBH promoter fragment (green) controlled by an 8× promoter (PRS2; brown), which confers selective expression in noradrenergic and adrenergic neurons.The vector contains four sequences coding for Mtr1a (light brown), the EGFP sequence protein (yellow), posttranscriptional regulatory element (WPRE) to enhance expression (red), the reporter gene SV40 promoter (no enhancer) (turquoise), the Class 1 integron ori (blue), the restriction enzyme pUC18-275 (dark blue), and the transcription regulator AmpR promoter-36 (amber).contrary, in all animals (n = 4), clear vMLC2-rh10 (EGFP = RED) transduction was present in the soma of NE-positive neurons within the LC (Fig. 6B).Next, we used these images for colocalization analyses applying Auto Threshold ImageJ plugin (Landini) and JACoP plugin option of the image analysis software Fiji.High correlation values (Fig. 6C) were found by Pearson's (PC; 0.68 ± 0.03) and overlap colocalization (OC; 0.93 ± 0.017).To evaluate the efficacy of the viral transduction, defined as the fraction of TH + cells coexpressing EGFP over the total number of TH + cells (Mander's coefficient M2), we compared the overlap of pixels between the pixels count of TH + cells with the pixels of EGFP + cells and found an overlap of ≥96% of the TH + cells as positively transduced.Similarly, we quantified the specificity (Mander's coefficient M1) of transgene (AAV-hrMT 1 ) expression as the fraction of EGFP + cells that coexpress TH + cells.Thereby, we found that ≥78% of the EGFP + cells are positively transduced in TH + cells (Fig. 6C).No damage to the LC given by the cannulation as well as the injection was observed (Fig. 6D).

Discussion
In this study, we investigated the effects of the selective MT 1 receptors partial agonist UCM871 on the vigilance states in rats, and we elucidated its underlying mechanism of action.UCM871, by selectively activating the MT 1 receptors, increased the duration of REMS during the 24 h light/dark cycle, mostly because of the effect of the drug on REMS during the light/inactive phase.Notably, we showed that the promotion of REMS is likely the result of the activation of MT 1 receptors localized in LC-NE neurons since (1) UCM871 inhibits LC-NE neuronal activity in a dose-dependent manner and through an MT 1 -mediated mechanism as its activity is blocked by the nonselective MT 1 /MT 2 receptor antagonist luzindole, but not by the selective MT 2 receptor antagonist 4P-PDOT; (2) the knockdown of melatonin MT 1 receptors in LC-NE neurons by a novel selective AAV vector nullifies the effect of UCM871 on REMS duration; and (3) no inhibitory effects of UCM871 on LC-NE neurons are present in rats with knocked-down MT 1 receptors in LC-NE neurons.
These data are consistent with our initial hypothesis based on previous results in MT 1 receptors knock-out mice.In those mice, we observed a significant reduction in REMS duration over the 24-hour sleep/wake cycle, primarily attributable to decreased REMS during the light/inactive phase (Comai et al., 2013).The LC is the primary source of NE projections throughout the central nervous system (Aston-Jones and Waterhouse, 2016) Figure 7.The knockdown of melatonin MT 1 receptors in LC-NE neurons abolishes the effects of UCM871 on REM sleep and on LC-NE firing activity.A, Cumulative analysis of the 24 h duration of the three vigilance states REMS (A.1), NREMS (A.2), and wakefulness (A.3) of transfected animals after the administration of UCM871 at 14 mg/kg (n = 10 rats) or vehicle (n = 8 rats).Duration of REMS (A.4), NREMS (A.5), or wakefulness (A.6) during the inactive/light phase in animals transfected with shMT 1 and treated with vehicle (n = 8 rats) or UCM871 at 14 mg/ kg (n = 10 rats).B, Schematic timeline of the electrophysiological experiment.C, Example of spontaneous firing rate histograms of LC-NE neurons after treatment with cumulative doses of UCM871 in rats transfected with control (yellow) and AAV-shMT 1 (green).D, Spontaneous firing rate of LC-NE neurons after administration of cumulative doses of UCM871 (3.5, 7, and 10.5 mg/kg) in rats transfected with control (yellow; n = 6, 1 neuron/rat) and AAV-shMT 1 (green; n = 6, 1 neuron/rat).E.1-5, Burst-firing activity parameters of LC-NE neurons transfected with control (yellow; n = 6, 1 neuron/rat) and AAV-shMT 1 (green; n = 6, 1 neuron/rat) following cumulative doses of UCM871.*p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle; # p < 0.05, ## p <0.01, ### p < 0.001, shMT 1 versus EGFP, by two-way ANOVA for repeated measures followed by Bonferroni's post hoc test.
and, as part of the ascending arousal pathway, is involved in the maintenance of the arousal state (Aston- Jones and Bloom, 1981;Ross and Van Bockstaele, 2021).Physiologically, the rate of spontaneous discharge of LC-NE neurons varies among the different vigilant stages, with higher activity during wakefulness, lower activity during NREMS, and virtually no activity during REMS (Aston- Jones and Bloom, 1981;Fuller et al., 2006;Aston-Jones and Waterhouse, 2016).Notably, we found that LC-NE neurons express MT 1 receptors in keeping with the data in mice by Klosen et al. (2019) showing scattered lightly labeled MT 1 -LacZ cells in the LC, and that the MT 1 receptor partial agonist UCM871 dose-dependently inhibits the activity of LC-NE neurons.Carter et al. (2010) demonstrated that while the optogenetic inactivation of LC-NE neurons in mice resulted in a decrease in both the overall wakefulness and the duration of individual awake episodes, optogenetic activation of LC-NE neurons instead induced immediate sleep-to-wake transitions, further implicating a causal role of LC in maintaining arousal.Interestingly, we reported that selective knockdown of MT 1 receptors in LC-NE neurons did not affect the duration of the three vigilance states, in agreement with previous studies showing no significant changes in the duration of vigilance states in animals where the NE synthesis was genetically inhibited (Hunsley and Palmiter, 2003) or the LC was pharmacologically lesioned (Lidbrink, 1974;Jones et al., 1977;Blanco-Centurion et al., 2007).Collectively, our data demonstrate that the activation of MT 1 receptors located in LC-NE neurons is sufficient for inducing REMS.However, since MT 1 receptors are also present in other brain regions implicated in sleep and projecting to the LC, such as the dorsal raphe nucleus, the suprachiasmatic nucleus (SCN), and the lateral hypothalamus (Lacoste et al., 2015;Sharma et al., 2018;Comai et al., 2019), they could be indirectly participating in the regulation of REMS.Indeed, the regulation of circadian activity of the LC comes from indirect projections from the SCN via the dorsomedial nucleus and paraventricular nucleus of the hypothalamus, as well as medial and ventrolateral preoptic areas (Aston- Jones et al., 2001;Fuller et al., 2006).Given that we administered UCM871 systemically, we cannot rule out the contribution of these or other areas of the brain to the observed effects of UCM871 on sleep and LC-NE activity.
Our group and others have previously demonstrated that selective activation of MT 2 receptors significantly promotes NREMS (Fisher and Sugden, 2009;Ochoa-Sanchez et al., 2011, 2014), while knocking out MT 2 receptors in mice significantly reduced the duration of NREMS (Comai et al., 2013).Collectively, these novel results further support the idea that the two melatonin receptors may modulate complementary or even opposite effects on sleep regulation (Gobbi and Comai, 2019a,b) and are a promising pharmacological target for developing new drugs (Liu et al., 2016).In keeping with this, nonselective MT 1 /MT 2 receptor agonists including melatonin itself or UCM793 have negligible effects on REMS or NREMS (Gobbi and Comai, 2019a,b).These data confirm the need to be selective rather than nonselective when targeting the melatonin system in neuropsychopharmacology.
This study shows that it is possible to activate REMS through a selective MT 1 receptor agonism in keeping with the role of the melatonin system in REMS.Indeed, according to Wang et al. (2011), melatonin in combination with clonazepam is the only medication indicated for REM sleep behavior disorder.Moreover, melatonin plays an important role in the impaired inhibitory neurotransmission, which is involved in the pathogenesis of REMS behavior disorder (Brooks and Peever, 2011).
It is important to mention that while activation of MT 2 receptors leads to a significant increase in NREMS paralleled by an increase in the power of δ band (Ochoa-Sanchez et al., 2011, 2014), activation of MT 1 receptors with UCM871 induces a significant increase of REMS paralleled by an increase in the power of θ band, which is a hallmark of REMS.Moreover, the dosedependent effects of UCM871 on the duration of REMS were also paralleled by an increase of δ and σ power in REMS but also an increase in δ and θ power in NREMS indicating that UCM871 may enhance the depth or intensity of sleep.Of interest, we found that the increase in the duration of REMS induced by UCM871 occurred without affecting significantly the overall architecture of sleep (bouts of REMS and NREMS as well as sleep fragmentation), as we observed only a minimal-but nonsignificant-increase in the number and duration of REMS bouts.This unique effect on the neural activity patterns and REMS dynamics deserves to be further investigated to understand their potential implications in sleep regulation and pharmacological application.
REMS has an important function in the mammalian brain since it mediates spatial and contextual memory consolidation (Boyce et al., 2017), in part due to the sparse activity of hippocampal adult-born neurons (Kumar et al., 2020) and activityand experience-dependent synaptic plasticity as well as experience-dependent synapse elimination (Zhou et al., 2020).Interestingly, both NREMS and REMS stages seem to play a complementary role in the process of learning (Tamaki et al., 2020), and their "flip-flop switch" is essential for human physiology (Lu et al., 2006).However, the mechanisms underlying the switch between NREMS and REMS stages remain largely unknown.In this context, we cannot rule out a role played by MT 1 and MT 2 receptors in the "flip-flop switch" between REM and NREM; indeed, they are differently expressed during the light/inactive phase (Pinato et al., 2017) and expressed in different brain regions modulating the sleep/wake cycle (Gobbi and Comai, 2019a,b).According to the "flip-flop switch" model, when LC-NE neurons become active, they inhibit the NREMS-promoting Figure 8. Effects of the administration of UCM871 (14 mg/kg) on the sleep/wake cycle of rats injected with the viral expression control protein (enhanced green fluorescent protein, EGFP).UCM871 (14 mg/kg) increases the duration of (A) REMS but not of (B) NREMS and decreases the duration of (C) wakefulness over 24 h.Similar effects of UCM871 (14 mg/kg) on (D) REMS, (E) NREMS, and (F) wakefulness are present also during the light phase.EGFP + vehicle = 6 rats; EGFP + UCM871 14 mg/kg = 5 rats.Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, Student's t test.
neurons, effectively "flipping the switch" from NREMS to wakefulness.Conversely, when they are less active, they allow REMS-promoting neurons to become more active (Lu et al., 2006;Dunmyre et al., 2014).Within this framework, serotonergic neurons of the dorsal raphe also inhibit NREMS-promoting neurons.When these 5-HT neurons become almost silent, they facilitate the activation of REMS-promoting neurons (Lu et al., 2006).
Interestingly, during REM sleep, the serotonin neurons of the dorsal raphe nucleus decrease their activity (Jones, 2005); in keeping, preliminary data showed that UCM871 acutely inhibits serotonergic neurons of the dorsal raphe nucleus (Comai et al., 2017).Consequently, a possible contribution of the dorsal raphe, which expresses MT 1 receptors (Lacoste et al., 2015), cannot be ruled out.
This study has some limitations: UCM871 was not tested in animals with impaired sleep and/or psychiatric disorders with comorbid sleep impairment or was only partially tested in female rats, where it decreased the LC-NE activity in a dose-dependent manner similar to males (unpublished data).Addressing these drawbacks will help move MT 1 compounds toward a possible clinical trial in humans, after toxicological studies.Despite these limitations, the present research demonstrates that selective activation of melatonin MT 1 receptors by UCM871 promotes REMS through inhibition of LC-NE neurons that express MT 1 receptors.These results add important novel insights to our current knowledge of the neurobiology of sleep.More importantly, they suggest that the MT 1 receptor may represent a novel selective pharmacological target for drug discovery in diseases characterized by REMS loss and impairments.Remarkably, REM Sleep Behavior Disorder (RBD) has been estimated to affect approximately 0.5-1% of the general population, representing a serious risk factor for the development of neurodegenerative disorders, including Parkinson's disease and other related conditions (Högl et al., 2022).While the precise role of MT 1 receptors in Parkinson's disease is still under investigation, some studies have suggested that melatonin via MT 1 receptors may exert neuroprotective effects while modulating circadian rhythms (Comai et al., 2019).Moreover, the lack of MT 1 receptors in transgenic mice not only impairs REMS, but generates a melancholic-like phenotype (Comai et al., 2015), and activation of MT 1 receptors improves a manic-like phenotype in mice (Tassan Mazzocco et al., 2023).Future research should address whether dysfunction of the MT 1 receptor may represent a common mechanism linking sleep, psychiatric disorders, and neurodegenerative diseases.
describe the experimental protocols.

Figure 3 .
Figure 3. Immunohistochemical expression of the MT 1 receptor in norepinephrine neurons of the LC.A, Schematical representation of the LC taken for the Western blot analysis.B, Immunoblotting with anti-MT 1 (1:200) and anti-α-tubulin (aT; 1:10,000) antibodies.Upper bands correspond to the positive blotting from the endogenous control α-tubulin protein whereas the lower bands correspond to the positive blotting from the MT 1 protein.MT 1 receptors decorated bands of approximately 40 kDa, at their expected molecular weight.The right panel displays the immunoblot with the immunogen peptide.Each lane represents the blotting from one rat.C, Immunoreactivity of MT 1 receptors in the LC, 5× (top) and 20× (bottom) show both sides of the LC nucleus expressing MT 1 receptors.Scale bar: 250 µm (top), 100 µm (bottom).D, Double immunofluorescence confirmed the expression of MT 1 receptors (red) in LC-NE neurons (TH, green).Sections were counterstained with DAPI (blue).Scale bar: 100 µm, 20 µm (expand).E, Percentage of immunoreactivity of neuronal colocalization (yellow), MT 1 cellular expression alone (red), and TH neuronal expression alone (green).

Figure 6 .
Figure 6.Transduction efficiency of the AAV-hrMT 1 viral vector is detectable in LC-NE TH + neurons but negligible in nucleus tractus solitarius (NTS), nucleus prepositus/medial vestibular nucleus (Pr/MVN), and periaqueductal grey (PAG) TH + neurons.A, Schematic timeline of the experiments and representative schema of coronal sections of the rat brain viewing the bilateral injection site (right) and histological section (left), showing the fluorescence of cell nuclei DAPI (blue) and endogenous tyrosine hydroxylase (TH, green) immunoreactivity.Scale bars: 100 and 20 µm in expand.B, Viral vector (shrMT 1 ) transduction after bilateral injection of 1 µl shrMT 1 in the LC was analyzed by confocal microscopy of coronal sections.Each row represents a different tissue section adjacent to the third ventricle, locus coeruleus (LC), nucleus tractus solitarius (NTS), nucleus prepositus/medial vestibular nucleus (Pr/MVN), and periaqueductal grey (PAG).The first column represents a cell nuclei DAPI (blue) fluorescence; the second column shows endogenous TH (green) immunoreactivity; the third column shows the shrMtnr1a transduction (GFP; red) immunoreactivity; the fourth column shows TH/GFP coexpression leveling cells; and the last column shows the magnification of the TH/GFP coexpression labeling cells.Structures that are positive for TH are indicated with a pink arrow, and structures positive for both TH/GFP are indicated with a white arrow.Scale bars: 100 µm in B Columns 1, 2, 3, and 4; and 20 µm in B Column 4. C, Thresholds for the images collected in B were acquired using the Auto Threshold ImageJ plugin (Landini), and Pearson's and Mander's coefficients were calculated with the JACoP plugin on the average of four animals.PC, Pearson's correlation; OC, overlap correlation; M1, Mander's overlap EGFP; M2, Mander's overlap TH.D, Immunoreactivity of TH in the LC, 5× (top) and 20× (bottom) after bilateral administration of 1 µl shrMT 1 confirming the integrity of the norepinephrine-expressing cells in the LC.Scale bars: 250 µm in C top; 100 µm in C bottom.E, Fold change detection in LC nucleus of animals transduced with virus expression control (EGFP; n = 5 rats) or the shRNA specific (shrMT 1 ; n = 5 rats).Data are reported as mean ± SEM. ***p < 0.001, Student's's t test (unpaired analysis).

Table 1 .
Number and duration (min)of REMS, NREMS, and wakefulness bouts during the 12 h light/dark phases after the subcutaneous treatment with UCM871 at the doses of 7, 14, and 21 mg/kg Data are reported as mean ± SEM.